nimbios investigative workshop individual-based ecology of microbes: observations and modeling
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NIMBioS Investigative Workshop Individual-Based Ecology of Microbes: Observations and Modeling June 8-10, 2011 Knoxville, Tennessee Q3: Interactions with the environment Elucidating the bacteria responsible for utilization of dissolved organic matter in streams Philips Akinwole. - PowerPoint PPT PresentationTRANSCRIPT
NIMBioS Investigative WorkshopIndividual-Based Ecology of Microbes: Observations
and ModelingJune 8-10, 2011
Knoxville, Tennessee
Q3: Interactions with the environment
Elucidating the bacteria responsible for utilization of dissolved organic matter in
streams Philips Akinwole
Personal Introduction
MSc in Environmental Science and Technology (2003-2005)International Institute for Infrastructural, Hydraulic, and Environmental Engineering, (UNESCO-IHE), Delft, the NetherlandsProject: Litter decomposition across multiple spatial scales in German stream networks
so…I’m broadly trained in stream and microbial ecology using stable isotope probing and phospholipid techniques (a multidisciplinary approach) to link microbial community structures to functions
… presentlyPhD Candidate in Microbial Ecology/ Department of Biological SciencesUniversity of Alabama, Tuscaloosa, AL (2007…)
… interested in linking microbial community structures to functionsPresenting Projects: Stable Isotope Probing of Dissolved Organic Matter Processing in StreamsSpatial Patterns of Microbial Signature Biomarkers in Stream NetworksMeasuring dissolved organic matter spiraling lengths: whole stream releases
Elucidating the bacteria responsible for utilization of dissolved organic matter in streams
Streams and rivers can be found everywhere. Bodies of flowing water moving in one direction from headwaters (springs, snowmelt or even lakes) and then travel all the way to their mouths, usually another water channel or the ocean. The characteristics of a river or stream change during the journey from the source to the mouth. Full of life; from microbes to plants (algae and macrophytes) and animals (fish and macroinvertebrates).
… some concepts
Dissolved organic matter (DOM), the largest pool of carbon in aquatic ecosystems, is heterogeneous in nature and supplies the needed energy and carbon to microbes; in streams it may meet the majority of heterotroph carbon demand (Kaplan et al., 2008).
…bacterial are the main heterotrophic microorganisms in stream responsible for the breakdown of organic biomass and the recycling of various key elements (N,P,S,C)
Neversink River, NY
Elucidating the bacteria responsible for utilization of dissolved organic matter in streams
One of the methods (biochemical and molecular) of studying microbial diversity/community structure is the Phospholipid Fatty Acid (PLFA) Analysis PLFAs are a major component of the cell membrane of all microbes (bacteria and eukaryotes) and they only remain intact in viable cells. are relatively easily extracted from natural samples. PLFA analysis as biomarkers provides direct information on the whole microbial community including quantitative information on viable (living) biomass, community structure (Population “Fingerprint”) and microbial activity. Fatty acids are identified and quantified by gas chromatograph (GC).
Elucidating the bacteria responsible for utilization of dissolved organic matter in streams
Stable Isotope AnalysisIsotopes are forms of an element that differ in the number of neutron (H, C, N, O & S)- functions as natural dyes/colors, generally tracking the circulation of elements (discrimination between heavy and lighter isotopes).-- trace ecological connections at many levels, from individual microbes to whole landscapes
Direct links between microbial identity and biogeochemical processes are now possible with stable isotope analysis of biomarkers (lipids, amino acids & nucleic acids)Usually, stable isotope ratios are given in the δ-notation,
which for carbon is defined as:
… measured with the gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS).
In stream ecosystems, monomeric constituents of DOM are most labile while polymeric /humic substances of terrestrial origin are the major constituents of DOM pool (40-70% of DOM in rivers) and have been considered biochemically refractory (Benner 2003)
… and poorly used by microbiota due to the apparent chemical recalcitrance of humic substances.
Elucidating the bacteria responsible for utilization of dissolved organic matter in streams
Recent studies (Frazier et al.2005, Kaplan et al., 2008 etc) have suggested the susceptibility of humic substances to microbiological and photolytic oxidations and could provide a supplement to heterotrophic metabolism.
This study was framed within the general question “Does streamwater DOM contribute significantly to stream ecosystem metabolism?” and particularly addressed the question “Which microorganisms within the stream community utilize humic-DOM?”
Elucidating the bacteria responsible for utilization of dissolved organic matter in streams
-generate a tracer model that is more reflective
of natural stream DOM, contrasting the use of
bicarbonate or acetate as tracers (e.g. Pace et
al., 2004; Johnson & Tank 2009),- we synthesized a 13C-DOM tracer from tulip
poplar tree-tissues with size and lability
fractions approximating streamwater DOM
(Wiegner et al., 2005).
…to do this
min24 26 28 30 32 34 36 38
pA
0
100
200
300
400
500
600
700
FID1 B, (CHYTRID1\RI.D)
24.126
26.706
28.568
28.814
29.244
30.335
30.481
30.772
30.906
31.066
31.545
32.553
32.620
32.741
32.940
33.207
33.300
33.805
35.286
36.816
36.899
39.427
Elucidating the bacteria responsible for utilization of dissolved organic matter in streams… research activities
Collection of streambed sediment from WCC
Sediments were incubated in mesocosm chambers with 175 µg C L-1 13C – labeled DOM
Microbial biomass and community structure were analyzed by PLFA
The fatty acid methyl esters (FAMEs) obtained from PLFA analyses were identified and quantified by GC, and analyzed for their δ13C values using the GC-IRMS.
For each experiment , treatments were identified as: T0 – sediments collected from WCC at the beginning of the experiments; T13C – sediments incubated for 48hrs in streamwater amended with 13C-labeled leachate and, T50 – sediments incubated for 48hrs in streamwater.
Four experiments were conducted in the light (unfiltered sunlight) and dark (chambers were covered with styrofoam and black plastic)
Results
Elucidating the bacteria responsible for utilization of dissolved organic matter in streams
br17:1a, a17, 18:1w5, i17, cy17:0, br18:0a, 10me16, i16, cy19, a15
22:6w3, 16:1w13t, 20:5w3, 16:3, 20:4w6, br18:0c, 14:0
16:1w7c br17:0, 16:4w1
17:0, 22:0, br19:1a, 20:0, 18:0, 18:1w9
PCA to evaluate changes in total microbial community structure during mesocosm incubation. Red arrows emphasize a comparison between T0 and T48/50 data (mesocosm controls) while blue arrows emphasize a comparison between T0 and T13C data (13C-DOM treatment). [● (Exp1), ♦ (Exp 2), ▲(Exp3), ■ (Exp4)]
Results
i15
a15
16 cl
uste
r*
16:0
10m
e16
cy17
,coel
uter
18 cl
uste
r*
cy19
20 cl
uste
r*
21:0
, 22:
6w3
-45
-40
-35
-30
-25T0/ Exp1 T48/Exp1 T13C/Exp1
PLFAs
δ13C
val
ue (
‰)
Elucidating the bacteria responsible for utilization of dissolved organic matter in streams
Stable carbon isotopic information for PLFAs from Experiment 1 (Dark) sediment incubated with and without 13C-labelled DOM
T0 = natural stream sediments T48 = natural steam sediments incubated with recirculating streamwater for 48 hrs T13C = natural steam sediments incubated with recirculating streamwater amended with 13C-DOM for 48hrs
FAME Bio marker EXP 1 (Dark)
EXP 2 (Light)
EXP 3 (Light)
EXP 4 (Light)
i15:0 Prokaryote √ √ √ √
a15:0 Prokaryote √ X √ √
16:0 Non specific √ √ √ √ 16:1w7c,
16:1w5c Prokaryote √ Depletion √ √
16:1w9 Non specific √ √ √ √
10me16:0 Prokaryote X X X X
cy17:0 Prokaryote √ X X X
18:2w6 Eukaryote X X ? X
18:3w3 Eukaryote √ X ? X 18:1w9c,
18:1w7c Non specific √ √ √ √
cy19:0 Prokaryote √ X X X
20:4w6 Eukaryote √ X ? ?
20:5w3 Eukaryote √ √ √ X
22:6w3 Eukaryote √ X X X
√= Enrichment X = No enrichment ? = Potential enrichment
Summary of incorporation of 13C-labeled DOM into microbial lipid by experiment
Results
Incorporation of label into PLFA was studied in light and dark incubations .Dark incorporation was fully sensitive to 13C-labelled DOM utilization
Conclusions
The PCA analysis of microbial biomass and community structure validate the robustness of mesocosm-based experimental design for determining the role of sediment microbes in processing streamwater DOM via incorporation of 13C-DOM into microbial fatty acids.
PLFA analysis indicated that both phototrophic and heterotrophic microeukaryotes were present in the sediment community. For the dark experiment, we assume that the 13C- labeled DOM uptake by prokaryotes is direct and not facilitated by photolysis.
The consistently labeled fatty acids: i15:0. 16:0, 16:1w9, 18:1w9c and 18:1w7c in our study are found in bacteria often associated with aerobic or facultative anaerobic metabolism (Findlay 2004).
Fatty acids 10me16:0 (showed no labeling) and cy17:0 (only showed moderately labeling when sediments were incorporated in the dark) are consistently found in bacteria associated with anaerobic metabolism, such as sulfate reducing bacteria.
Eukaryotic marker fatty acids (20:4w6, 20:5w3) were most constantly labeled in the dark experiment suggesting that microeukaryotic predators are consuming bacteria utilizing 13C-labeled DOM.
… motivation for and challenges in individual base approach
Individual based approach use information at the level of individual organisms and derive characteristics of the population as an integral of all individuals represented in the population.
- Successfully applied in conservation biology…however, they include many parameters which are difficult to estimate for natural populations and which rely on very detailed knowledge on the species and the spatial structure of its populations.
- I’m interested in how to apply/use individual base model in my work, - advances made in microbial ecology, - applications and uses in other areas
Thank you